J . gen. Mimbiol. (1965), 40,227-234
With 1 plate
Printed in meat Britain
227
The Stability of Mycoplasma mycoides
BY A. W. RODWELL
Divisio?~
of A n i w l Health, Animal Health Research Laboratory
C.S.I.R.O., Parhwilb, N . 2, Victoria, Australia
(Received 24 February 1965)
The morphology of Mycoplasma mycoides was well preserved after
washing and suspension in buffered 0-4M-sucrose solutions, but the
survival of viable particles was no better, and the loss of ultraviolet (u.v.)absorbing substances and the decrease of turbidity was no less than in
hypotonic solution (0.01 M-tris HCl or 0.01 wNa,HPO KH4P04).The
addition of Mg2+, Ca2+, spermidine or spermine (increasing order of
activity) decreased the decrease of turbidity and loss of u.v-absorbing substances. Ca2+and Mg2+,but not spermine, increased the degree of survival
of viable particles. Ethylenediaminetetra-acetate (EDTA; 0.01 M) increased the loss of u.v.-absorbing substances, and decreased the turbidity
and degree of survival. Ca2+, Mg2+and spermine annulled the effects of
EDTA on loss of u.v.-absorbing material and on the turbidity, but only
Cae+prevented the lethal effect of EDTA. Filaments disappeared and cell
volume increased when the organisms were transferred from hypertonic
to hypotonic solutions; the shape changes were reversible.
+
INTRODUCTION
Smith & Sasaki (1958) showed that the viability of some Mycoplasma strains was
much less affected by the osmotic pressure of the suspending medium than had been
supposed. Butler & Knight (1960) confirmed this observation and showed also that
metd-chelating agents (diethyldithiocarbamate, ethylenediaminetetra-acetate, 8hydroxyquinoline), manganese dioxide and the use of de-ionized water of high
quality increased the degree of survival in suspensions in dilute buffer solutions. In
contrast, Ca2+ ions have a marked stabilizing effect on bacterial spheroplast suspensions in hypotonic solutions (Tabor, 1962) and spermine, spermidine, streptomycin
and polylysine were very effective stabilizers, while monovalent cations, Mg2+ and
the diamines 1&diaminobutane and 1,5-diaminopentanewere relatively ineffective.
Razin & Argaman (1963) compared the susceptibility of mycoplasmas, bacterial
L-forms, spheroplasts and protoplasts to lysis by osmotic shock, by alternate
freezing and thawing and by surface-active compounds. The mycoplasmas and
bacterial L-forms were more resistant to osmotic shock than were the bacterial
protoplasts and spheroplasts but, like the protoplasts, the mycoplasmas were very
sensitive to lysis by surface-active substances. Mycoplasma strains differed in
their sensitivity to lysis by osmotic pressure changes, strains of Mycoplasma
rnycoides being among the more resistant ones (Razin, 1963). Razin (1964) showed
that the Mycoplasma strains he examined resisted osmotic shock a t Oo, but underwent rapid lysis at 37'. Divalent and polyvalent cations, in concentrations as low
as
M, protected Mycoplamnu luidluwii from osmotic lysis.
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228
A. W. RODWELL
METHODS
Organisms. The strain v5 of Mycoplamnu mycoides and the strain N of M. Capri
were used. The latter strain has been found not to form detectable amounts of polysaccharide (Plackett, Buttery & Cottew, 1963). The organisms were grown in
supplemented BVF-OS medium (Plackett et al. 1963) and harvested when the
turbidity of the cultures had increased to between one quarter and three quarters
of the maximum expected value. Within this turbidity range, the organisms were
highly filamentous and the ratio of colony count of viable particles to turbidity was
approximately constant.
Reagents. The salts used were of AR quality; the water was glass distilled, but no
special precautions were taken to prevent metal ion contamination. Spermine
(grade B) and spermidine (grade A) were obtained from the California Foundation
for Biochemical Research, and 1,4-diaminobutane from E. Light and Co. (Colnbrook, Bucks., England). Sucrose (M solution) was freed from cations by passage
through Dowex 50 (Hf) column. The solutions containing ethylenediaminetetraacetate and divalent metal ions were adjusted to pH 7.4 with NaOH.
Procedure. Ten-ml. volumes of culture were chilled, and centrifuged at 15,000
rev./min. for 20min. (Spinco, model L, rotor 40). The supernatant fluids were
poured off and the walls of the tubes wiped dry with filter paper. The deposits of
organisms were then washed twice by resuspension in 10-ml. volumes of the test
solutions. The entire process, which occupied about 3.5 hr, was performed in the
cold.
Counts of viable particles determinations were made by a plate colony count
method after the suspensions had been kept about 1hr at 0-2", and again in most
experiments after a further period of 1-3 days at 2". The results are expressed as the
% of the colony count of the original culture. Turbidity measurements were made
at 650 mp (Beckman Model DU, 1cm. light path) on samples of the suspensions
(warmed to room temperature) at the end of the storage period, and are expressed
as yo of the turbidity of the original culture. Leakage of u.v.-absorbing material
was measured at 250 mp in both of the supernatant fluids after washing, and also
in the supernatant fluid obtained after centrifuging a sample of the suspension after
storage. The sum of these measurements, corrected where necessary for the extinction of the washing fluid, are expressed as yo of the extinction of an alkali lysate.
For this, the unwashed pellet from 10 ml. culture was incubated at 37" with 1 ml.
N-NaOH for 1hr and the lysate diluted to 50 ml. for measurement.
The volumes of packed cell pellets were measured after centrifugation in graduated
capillary-tipped tubes at 18508 for 18 hr at 2". Polyvinylpyrrolidone (1 yo,w/v,
in the suspending fluid) was used as an indicator for measuring the intercellular
volume (Wetherell & Pollack, 1962).
Electron microscopy. Nine volumes of a solution of the same composition as that
of the suspending fluid but containing in addition either 4 % (w/v) formaldehyde or
5 % (w/v) glutaraldehyde were added to 1 vol. of suspension of organisms. Phosphate buffer was used in the suspending fluid to avoid pH changes. After standing
overnight at 2 O , the organisms were washed and suspended in water. Droplets were
air-dried on electron microscope grids before metal-shadowing. No differences in
morphology between material fixed in formaldehyde or glutaraldehyde were
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Stability of mycoplama
229
detected, but the impression was formed from dark field examination in a light
microscope that both fixatives resulted in some shrinkage, and in an increase in the
number of beaded filaments.
RESULTS
Efects on turbidity, loss of ultraviolet-absorbing material and survival
Mg2+,Ca2+arnd EDTA. There was a decrease in turbidity, a loss of u.v.-absorbing
material from the organisms and a decrease in the colony count after washing the
organisms in dilute tris or phosphate buffers (0.01 M, pH 7.4). The magnitude of
Table 1. The eflect of Mg2+ and Ca2+on the stability of Mycoplamna rnycoides
Expt.
1
2
*
Divalent cation salts*
(M concn.)
Turbidity
(
None
MgSO, (0.001)
MgSO, (0.01)
MgSO, (0.1)
None
CaCl, (0401)
CaCl, (0.01)
CaCl, (0.1)
Salts added to 0.01 M - t r h HCl (pH 74).
yo of alkali lysate.
%)t
Extinction
at 260 m p
50
78
84
95
Viable count (%) after
stated period a t 0-2'
,A
--r
( %)t
28
24
25
70 hr
13
15
0.3
0-2
21
41
60
57
49
57
4'4
45
35
27
20
23
60
70
87
88
1 hr
t Percentage of culture.
8
2-5
27
19
$ Sum of 260 mp extinc-
tion of washings as
Table 2 . The eflect of Mg2+ and Ca2+and EDTA on the stability of
Mycoplasma mycoides
Expt.
1
2
Additions*
(M concn.)
None
EDTA (0.001)
EDTA (0.01)
MgSO, (0.01)
EDTA (0.001);MgSO, (0.001)
EDTA (0.01); MgSO, (002)
EDTA (0.01)
MgSO, (0.01)
EDTA (0.01); MgSO, (0.02)
CaCl, (0.01)
EDTA (0.01); CaCl, (0.02)
MgSO, (0.01);CaCl, (0-01)
EDTA (0.01); MgSO, (0.01)
CaCl, (0-02)
Turbidity
(%It
73
71
60
89
89
82
35
83
80
97
83
98
83
Extinction
at26Omp
(%)$
Viable count (yo)after
stated period a t 0-2"
37
50
55
29
29
32
81
27
33
%
28
22
28
A
-l
lhr
70 hr
56
56
2.0
65
48
8
1.0
48
22
47
28
48
31
21
23
0.01
59
45
0.8
0.2
22
2.1
32
22
34
24
* Additions to 0.01 ~ t r i s H C 1(pH7.4). -f Percentage of turbidity of culture.
260 mp extinction of washings as yo of alkali lysate.
2 Sum of
these changes was about the same for either buffer. Tris buffer was used for most of
the experiments to be described. The effect of pH value was not investigated. The
addition of MgSO, or CaCI, in concentrations of 0.01 M or higher decreased these
G. Microb. XL
15
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A. W. RODWELL
230
changes (Table l), whereas ethylenediaminetetra-acetic acid (EDTA) at 0.01 M
increased them, its effect on the loss of viability being greater after storage (Table 2).
At one-tenth this concentration, EDTA had no effect on survival, but increased
the leakage of u.v.-absorbing material. Mg2+(0.01g. ions/l.) prevented almost
completely the decrease of turbidity, and the loss of u.v.-absorbing material which
Table 3. The effect of polyamilzes on the stability of Mycoplama mycoides
Polyamhe*
(M concn.)
None
Spermine (0-00001)
Spennine (0.0001)
Spermine (0.001)
Spermhe (0.005)
Spermidine (0.OOOl)
Spermidine (0.001)
Spermidhe (0.005)
1,4-Diaminobutane(0.005)
Turbidity
97
Extinction a t
26omP (%I$
21.4
18.7
13-5
11.7
11.7
17.0
13.4
13.3
83
19.5
(%It
71
80
86
89
95
78
87
* Added to (0.01 M tris HC1 (pH 7.4). t Percentage of turbidity of culture. $ Sum of 260 mp
extinction of washings as yo of alkali lysste.
Table 4. The eflect of MgZf, Ca2+ and spermine on the stability of
Mycoplama mycoida
Expt.
1
2
3
Cation additions
(M concn.)
None
MgS04 (0.01)
CaC1, (0.01)
Spermine (0.001)
None
MgSO, (0.01)
CaCl, (0.01)
Spermine (0.001)
None
MgSO, (0.01)
Spermine (0.001)
MgSO, ( O * O l ) t
Spermine (0-001)
Extinction
Turbidity at 260 mp
(%It
(%H
59
35
19
17
14
32
20
16
15
30
24
18
17
79
87
82
65
90
92
96
'76
88
95
104
Viable count (yo)after
stated period at 0 - 2 O
I
2 hr
21
39
21
20
42
75
41
50
41
60
49
38
A
24hr
11
43
25
3
19
81
47
10
-
-
1
48 hr
11
70
41
4
12
28
6
32
* Added t o 0.01 M t r i s HCl (pH 7.4). 'f Percentage of turbidity of culture. $ Sum of 260 mp
extinction of washings as yo of alkali lysate.
occurred in the presence of 0.01 M-EDTA alone, but only partly prevented the
effect of EDTA (0.01 M) on survival (Table 2). Ca2+(0*Olgions/l.) almost completely prevented the lethal effect of EDTA (0.01M) as well as preventing effects on
turbidity and on loss of u.v.-absorbing material (Table 2).
Polyamines. Spermine (0.0001 M) had about the same effect as 0.01 M-Mgso, or
0.01 M-C~CI, on turbidity and the loss of u.v.-absorbing material. Spermidine
(0.001 M) had about the same effect as 0.0001 M-spermine; 1,4-diaminobutane had
little effect at 0.005 M (Table 3). Spermine, in contrast to MgSO, and CaCl,, did not
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Stability of mywplasma
231
increase the survival of viable particles during storage, although the organisms
survived in its presence when MgSO, was also added (Table 4). Spermine (0.001 M)
was as effective as Ca2+(0.01g. ions/].) in preventing the effects of 0.01 M-EDTAon
the loss of u.v.-absorbing material and on turbidity (Table 5).
Table 5 . The efect of Caa+,EDTA and spermine on the stability of
Mycoplasma mycoides
Tris = 0.01 M-tris HCl (pH 7-4); P = 0.01 M-N~,HPO,+KH,PO, (pH 7.4); SUC= 0-4 Msucrose.
Composition
Turbidity
Extinction a t
(M concn.)
t %)*
260mtc (%It
Tris + CaCl, (0.01)
93
20
Tris +EDTA (0-01)
55
43
Tris +EDTA (0.01)+CaCl, (0-02)
80
23
Tris +EDTA (O.Ol)+spermine (0.001)
76
23
P +EDTA (0-01)
49
43
P+EDTA (O-Ol)+spermine(0.001)
75
24
P + SUC+EDTA (0.01)
34l
51
P + SUC+ EDTA (0.01) + spermine (0.001)
73
21
* Percentage of turbidity of culture.
alkali lysate.
t
Sum of 260mp extinction of washings as % of
Table 6. The e$ect of sucrose, NaCl and KC1 on the stability of Mycoplasma mycoides
*
Added
solute
(Mconcn.)*
Total solute
concn.
(mold)
None
Sucrose (0.25)
Sucrose (0.4)
NaCl (0.15)
NaCl (0.25)
KCl (0.15)
KCl (0.25)
0.03
0-30
0.50
0.30
0.50
0.30
0-50
Extinction
at 260 mp
Turbidity+
(%I$
Viable count
(yo)after
1hr at 0"
78
67
61
65
65
70
61
22
30
25
35
35
34
33
45
32
36
42
30
58
30
Added to 0.01 M - t r i s HCl+0.01 rd-MgSO,. t Percentage of turbidity of culture. 2 Sum of
yo of alkali lysate.
260 mp extinction of washings as
Tonicity. In the absence of stabilizing cations, the decrease in turbidity and the
loss of u.v.-absorbing material were greater when the organisms were washed and
suspended in solutions containing sucrose (0.4 M) than they were in hypotonic
solutions. In the presence of stabilizing cations the addition of sucrose made little
difference. The effects of adding sucrose ( 0 . 2 5 ~ 0, . 4 ~ )and of NaCl and KC1
(0.15M and 0-25 M) to tris+MgSO, (0.01 M) solution are shown in Table 6. The
decrease in turbidity and the loss of u.v.-absorbing material were slightly greater,
and survival about the same, in the presence of these solutes as they were in the
suspension in hypotonic solution.
Effects on morphology
Observations were made by dark field microscopy and by electron microscopy of
formaldehyde-fixed or glutaraldehyde-fixed suspensions of Mycoplasma mycoides.
The cultures examined contained a high proportion of filamentous organisms, but
15-2
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A. W. RODWELL
232
other forms were also present. The filaments were preserved after washing and
fixing in solutions having a total solute concentration of 0-3or 0.5 mold (dilutebuffer +sucrose or NaC1) followed by washing in water. There were no filaments in
suspensions of organisms washed and fixed in hypotonic (0.03 molal) solutions.
Electron micrographs of these suspensions showed electron-dense almost spherical
forms, flattened forms and other material. The addition of stabilizing cations or of
EDTA had no marked effects on the morphology of the organisms when suspended
either in sucrose (0-5 molal) or in hypotonic (0.03 molal) solutions.
Table 7. Eflect of tonicity of suspending medium on the packed cell
volume of Mycoplasma strailzs v5 and N
Organisms were washed and suspended in 0-5 molal solution. 1 vol. suspension was
added to 10 vol. solution of molality 0.03 or 0.5, and the suspensions were then centrifuged in graduated capillary-tipped tubes at 1850g for 18 hr at 2'. The cell volumes
recorded are the pellet volumes from which the intercellular volumes (as determined
by the amount of PVP in the pellets) were subtracted.
Strain
v5
v5
N
Age of culture Mold solute
(hr)
concentration*
40
0.07
0.50
18
0.07
0.50
18
0.07
0.50
Cell vol.
(Pl.)
41
23
18
8.5
9.6
5.4
+
* Compositionof solution: 0.01 m-tris HCI (pH 7.4). +0.01 M-MgSO, 1 yow/v polyvinylpyrro+sucrose 0-02 M (total solute concentration = 0.07 molal), or 0.4 M (total solute
lidone (PVP)
concentration = 060 molal).
The effects of the tonicity of the suspending fluid on morphology, and the reversibility of these changes, are illustrated in P1. 1, figs. 1 3 ; figs. l a and l b show the
organisms after they had been washed and resuspended in 0-01M-phosphate +0.01 M
MgSO,+ 0.001 M-spermine+0.4 M-sucrose solution (hypertonic solution); figs. 2 a
and 2 b show their appearance after they had been washed in the hypertonic solution
and then suspended in a solution of the same composition but lacking sucrose
(hypotonic solution); figs. 3a and 3 b show their appearance after they had been
suspended successively in hypertonic, hypotonic and again hypertonic solutions.
The flattened forms seen in P1. 1, figs. 2a and 2b, may have been spherical in
suspension and have collapsed during drying on the grids. Dark field examination
of the unfixed suspension in a light microscope showed only what appeared to be
spherical forms of various diameters with a maximum of about 1p. The rapidity of
the shape changes was evident from the complete absence of streaming birefringence
in the suspension in hypotonic solution and its immediate reappearance when the
organisms were resuspended in hypertonic solutions. Measurements of cell volumes
by using polyvinylpyrrolidone to measure the volume of the intercellular space in
the cell pellets are shown in Table 7. Organisms were harvested from an exponential
phase culture of Mycoplasma mycoides strain v 5 which contained numerous filaments, from a stationary phase culture of v5 strain which did not contain filaments,
and from an exponential phase culture of the M. Capri strain N which consisted
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Stability of mycoplasma
233
mostly of short rod-like forms. The cell volumes were, in each case, about twice as
great when the organisms were suspended in hypotonic solution as when in hypertonic solution.
DISCUSSION
These observations support those of others (Smith & Sasaki, 1958; Butler &
Knight, 1960 ; Razin, 1963) that Mycoplasma organisms survive well in hypotonic
solutions. They differ from those reported by Butler & Knight (1960) who found
that, within an optimal concentration range, metal ehelating agents, including
EDTA, increased survival. In the experiments reported here, EDTA decreased
survival and turbidity and increased the loss of u.v.-absorbing material, whereas
Ca2+and Mg2+had a stabilizing effect. These conflicting results may be due to differences in concentrations of toxic metal ion impurities. The stabilizing effects of
divalent cations and of polyamines for Mycoplasma mycoides are similar to those
described by Tabor (1962) for bacterial protoplasts and spheroplasts and by Razin
(1964) for M. ZuidZazvii. Razin found that any of a series of divalent and trivalent
cations as well as spermine and spermidine at 10-5 M and even lower concentrations,
prevented lysis during incubation of suspensions of M . ZadZawii at 37'. Spermine
at
M also prevented death in a hypotonic medium. I n the experiments reported
here, spermine did not prevent death of M. mycoides. The observed interactions
between Ca2+,Mg2+,spermine and EDTA may be the result of a competition between
EDTA and the cell membrane for Ca2+ and Mg2+ on the one hand, and between
Ca2+,Mg2f and spermine for common binding sites on the membrane on the other.
Mycoplasma mycoides responds to changes in the tonicity of the suspending
solution by changes in shape and volume. The volume changes for individual undamaged organisms may be larger than those measured for the whole suspensions,
because many of the organisms may have been damaged and their property of
responding to osmotic pressure changes destroyed during the manipulations. In the
case of the highly filamentous organisms of the v 5 strain harvested from the 18 hr
culture, the volume changes may have been considerably larger than the measurements shown in Table 7 for another reason. M. mycoides synthesizes a large amount
of polysaccharide which is believed to form an extracellular capsular or slime layer
closely surrounding the organisms in young cultures and later shed into the medium
(Plackett et al. 1963). The suspension of organisms harvested from the younger
culture would therefore contain a much larger amount of this extracellular material.
The ratio of the volume to the number of turbidity units in the samples centrifuged
was about four-fold greater for the suspension from the 18 hr culture than it was for
the suspension from the 40 hr culture. The filaments have a very large ratio of surface area to volume. If a filament 0.1 ,u in diameter and 7,u in length and bounded
by a membrane 85 A in thickness were transformed to a sphere of the same surface
area, the volumes within the external and internal surfaces of the membrane would
increase by factors of 5.5 and 8, respectively; the diameter of the sphere would be
0 . 8 ~This
.
is about the diameter of the larger forms observed by dark field examination in a light microscope in unfixed suspensions in hypotonic solution. The plastic
filamentous mycoplasmas therefore have the potential to increase their volume
several-fold by dilution of their contents (that is to decrease their-inernal ostmotic
pressure) without an increase in surface area. This may help to explain their
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A. W. RODWELL
234
relative tolerance to osmotic pressure changes compared with bacterial protoplasts,
whose volume, within a narrower tonicity range, is inversely related to the osmotic
pressure of the medium (Weibull, 1955) but which undergo lysis in dilute solutions.
Razin (1963) found that the sensitivity of certain Mycoplasma strains to osmotic
lysis decreased with the age of the culture. The older cultures would contain few
filaments. Changes in size, shape, lipid composition of membrane, and internal
osmotic pressure are doubtless important also in determining stability. AU of these
may change with age of culture.
I am indebted to Mr A. Abbot of the Walter and Eliza Hall Institute of Medical
Research for the electron micrographs.
REEXRENCES
BUTLER,
M. & KNIGHT,
B. C. J. G. (1960). The survival of washed suspensions of mycoplasma. J. gen. Mimobiol. 22,470.
& COTTEW, G.S. (1963). Carbohydrates of some Mycoplasma strains. Recent Progress in Microbiobgg: Synp. 8 int. Congr. Mierobiol. p. 535.
RAZIN,S. (1963). Osmotic lysis of Mycoplasma. J . gen. Microtn'ol.33,471.
RAZIN,S. (1964). Factors influencing osmotic fragility of Mycoplasma. J. gen. Microbiol.
36,451.
RAZIN,
S.& ARGAMAN,
M.(1963). Lysis of Mycoplasma,bacterial protoplasts and L-forms
by various agents. J. gen. Miclobiol. 30, 155.
SMITH,P.F. & SASAKI,
S. (1958). Stability of pleuropneumonia-like organisms to some
physical factors. Appl. Microbiol. 6,184.
TABOR,
C. W. (1962). Stabilization of protoplasts and spheroplasts by spermine and other
polyamines. J . Bmt. 83, 1101.
WEIBULL,C. (1955). Osmotic properties of protoplasts of Bacillus mgatherium. Exp.
Cell. Res. 9,294.
WETHERELL,
D.F. & POLLACK,
J. D. (1962). Polyvinylpyrrolidone as an indicator for
measuring intercellular space in packed cell pellets. J . B a t . 84, 191.
PIACKETT,
P., BUTTERY,
S. H.
EXPLANATION OF PLATE
Figs. 1-3 illustrate the effect of the tonicity of the suspending medium on the shape of Mycqlasma
mycoides organisms. Figs. la, b are of organisms washed and suspended in a solution of 0.01Mphosphate (pH 7.4) 0.01 na-MgSO,+ 0.001w-spermine 0.4 nf-sucrose (hypertonic solution). Figs.
2a, b are of organisms washed in hypertonic solution and then suspended in a solution of the same
composition but lacking sucrose (hypotonic solution). Figs. 8a, b are of organisms suspended
successively in hypertonic, hypotonic and then hypertonic solutions. The organisms for figs. 1-8
were then fixed for 18 hr at 0' in solutions of the same composition but containing 5 % (w/v)
glutaraldehyde, and washed and suspended in water. Gold-palladium shadowed. Figs. 1 a, 2 a and
3a, ~1500;
figs. lb, 2 b and 3b, x8600.
+
+
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Journal of General Microbiology, Vol. 40, No. 2
A. 1)’. RODWELL
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Plate 1
(Facing p . 234)